An Outbreak of ST859-K19 Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae in a Chinese Teaching Hospital

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has been increasingly reported worldwide. Here, we report an outbreak caused by sequence type 859-K19 (ST859-K19) CR-hvKP isolates in a teaching hospital in China. Interestingly, K. pneumoniae ST859 was a single-locus variant of ST11 but has never been reported before. A total of 11 nonrepetitive ST859 CR-hvKP isolates were collected from 11 patients, 3 of which died of severe CR-hvKP infection. Antimicrobial susceptibility assay results showed that all the 11 CR-hvKP isolates exhibited high-level resistance to commonly used antibiotics, only remaining susceptible to colistin, tigecycline, and ceftazidime/avibactam. Whole-genome sequencing (WGS) was performed using the Illumina platform for the 11 CR-hvKP isolates, and RJ9299 was further sequenced using the PacBio platform. A phylogram tree using WGS data revealed that all the 11 CR-hvKP isolates were clustered in 1 clade, which probably indicated clone transmission. Determinants of resistance and virulence gene analysis using WGS data confirmed the 11 isolates had almost identical resistance gene profiles (blaKPC-2, blaTEM-1B, blaSHV-187, rmtB, fosA6) and virulence gene (rmpA, rmpA2, iucABCDiutA) profiles, which hint at clone spread again. The complete genome size of RJ9299 was 5,875 kb, including a 5,445-kb chromosome, a 215-kb virulence plasmid (pVir-CR-hvKP-RJ9299), a 109-kb blaKPC-2-harboring plasmid (pKPC-2-RJ9299), and three circular plasmids. Comparative genomics showed pVir-RJ9299 (IncHI1B type) and pKPC-2-RJ9299 (IncFII-IncR) possessed over 99% similarity to pLVPK and pKPC-CR-hvKP-C789, respectively. Serum resistance assays and a Galleria mellonella infection model showed the 11 isolates exhibited different levels of virulence. This is the first report of an outbreak caused by ST859 CR-hvKP isolates. IMPORTANCE The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) in China has posed a great threat to public health, especially in the highly transmissible ST11 clone. With the transmission of virulence and resistance determinants, CR-hvKP isolates have been reported in an increasing number of sequence types (STs), including ST23, ST65, ST1797, ST43, ST231, ST147, ST15, ST383, ST268, ST595, ST375, ST48, and ST307. Here, we report an outbreak caused by ST859-K19 CR-hvKP isolates in a teaching hospital in China. ST859 is a single-locus variant of ST11. There is no literature on ST859 K. pneumoniae in public databases, let alone ST859 CR-hvKP isolates. To our knowledge, this is the first report to depict the molecular and genomic characteristics of ST859 CR-hvKP isolates. Active surveillance approaches should be implemented to promptly find the spread of CR-hvKP isolates in health care settings.

Minor comments: 1. The authors identified the CR-hvKP with positive string test, so the first sentence "All eleven CR-hvKP outbreak isolates had a hypermucoviscous phenotype with positive string test" is not necessary. 2. Figure 1: I cannot find "another two isolates were respectively from blood and drainage" in the figure 1. Please correct it. 3. Figure 2: "The eleven ST859-K19 CR-hvKP isolates were clustered into clade I. Six ST11-K64 KPC-2 producing isolates which had positive string test, namely CR-hvKP, were clustered into clade III. Nine ST11-K64 KPC-2 producing isolates which had negative string test, namely CRKP, were clustered into clade II." I think the figure 2A had a mistake, and please clarify it. 4. Compared to pKPC-CR-hvKP-C789, RJ9299 had a deletion, please annotate the deletion region.

Reviewer #2 (Comments for the Author):
This is a description of an outbreak of K. pneumoniae ST859 (which is a single locus variant (SLV) of ST11) carrying a virulence plasmid and blaKPC-2, the latter in a separate plasmid to the former, among 11 patients, several of whom unfortunately died. For me, I find the insistence on this being novel because it is a new ST (it is an SLV of ST11, with probably only a single nucleotide change in the tonB gene) is overplayed and I would always exercise caution in describing an account as the first description. The other thing that really strikes me about this is that the virulence plasmid is so like pLVPK, which is a nonconjugative virulence plasmid associated with hypervirulent K2-CG43. So how did it get into this type? Usually, where you find virulence plasmids in 'high-risk' clones such as ST11, ST15, ST147 etc., they are hybrid virulence/resistance plasmids that have become conjugative through recombination (see for example Xie et al., 2020 A hybrid plasmid formed by recombination of a virulence plasmid and a resistance plasmid in Klebsiella pneumoniae. J Glob Antimicrob Resist 23:466-470), which is actually a more frightening prospect -with a non-conjugative plasmid the virulence plasmid is at least confined to that type. Maybe you could discuss this? I worry about your defining isolates as hypervirulent on the basis of the string test -it is at best just an indication. There is no mention of how you did the nanopore sequencing; also did you use the fully assembled sequence of RJ9299 as reference, or just the Illumina sequence? Is the sequence of RJ9299 on GenBank (or similar) -if not, it needs to be and an accession number provided. Other specific points 'CR-hvKP has been reported in an increasing number of STs, including ST23, ST65, ST1797, ST43, ST231 and ST307' There are quite as few more -like ST147, ST15, ST383... The following review paper may be helpful to include Yang X, Dong N, Chan EW, Zhang R, Chen S. Carbapenem Resistance-Encoding and Virulence-Encoding Conjugative Plasmids in Klebsiella pneumoniae. Trends Microbiol. 2021 Jan;29(1):65-83. 'while ST258 was the main prevalent clone in North America, Latin America, and several European countries (6-9). ' Can't help but think that this is outdated and overplayed -it certainly is not my experience in the UK. 'All the patients received antimicrobial treatment, including carbapenem alone or in the combination with other alternative antibiotics when necessary' Why were patients treated with carbapenem alone for a carbapenemase producing organism? fosA is intrinsic in K. pneumoniae 'In terms of virulence genes, they all carried aerobactin (iucABCDiutA) and salmochelin (iroE) regulator of mucoid phenotype A (rmpA and rmpA2)' -but the virulence plasmid carried iroBCDN; surely that should be mentioned here. Not sure about this chromosomal iroE. Number of SNPs -please say how many bases were compared.

Reviewer #3 (Comments for the Author):
This study is the first to report the prevalence and dissemination of ST859 CR-hvKp strains. The characteristics of 11 CR-hvKp strains were analyzed by DNA sequencing, and genome-wide analysis was performed, which has important reference value for clinical anti-infection treatment and nosocomial infection control. I have some comments as follows. 1. The MIC data in Table 1 is the exact MIC value, or has the prefix "{less than or equal to}" or "{greater than or equal to}"? which is inconsistent with the description of the data in the text. In the text, it shows "{greater than or equal to}64", "{greater than or equal to}128" and "{less than or equal to} 0.25" etc. For example, not all 11 strains of imipenem have a MIC of "{greater than or equal to}64", and the MIC range should be 32-{greater than or equal to}128 mg/L according to table 1.

Reviewer #1
1. In this study, so many isolates were collected from one patient, for example, RJ9229 had another 17 isolates from sputum, and only the first isolate was considered as CR-hvKP. How about the following isolates, and so as the other patients?
Among the eleven patients, there were three patients from whom we also collected another CR-hvKP isolate besides CR-hvKP in our study. RJ9950 had a ST859 CR-hvKP after RJ9950 isolate, RJ10129 had a ST412 CR-hvKP isolate before RJ10129 isolate. RJ9690 had a ST218 CR-hvKP isolate and a ST 859 CR-hvKP isolate before and after RJ9690. These information has added to Fig1. The other eight patients had no another CR-hvKP isolates before and after CR-hvKP in our study.
2. Lots of isolates were isolated from sputum, and the underlying diseases of patients included hypertension, cerebral hemorrhage and tumor. Since hvKP usually caused liver abscess, endophthalmitis, meningitis, and metastatic infectious diseases. How about your definition of hvKP, please discuss in the manuscript?
Until today, there is no unified and standard definition of hvKP. In the present study, string test was phenotypically used to screen hvKP, which was finally defined by aerobactin production. CR-hvKP was defined by both aerobactin production and resistance to any of the carbapenems. Please see the MATERIALS AND METHODS section which marked in blue in "Marked-Up Manuscript ".
3. The analysis of Illumina and nanopore sequencing was different in this study, for example, in nanopore sequencing analysis, fimbriae, enterobactin, and yersiniabactin were considered as virulence factors, but not in Illumina sequencing analysis.
Sorry, we used PacBio platform, not nanopore for RJ9299 sequencing. We have corrected it. In fact, the analysis of Illumina and PacBio sequencing was same. I only wrote some important virulence factors in the eleven isolates sequenced by Illumina platform. Now, I have added all the virulence factors in the "Marked-Up Manuscript ". Please see the RESULTS section (Resistance genes, virulence genes profiles and phylogenetic analysis) which marked in blue in "Marked-Up Manuscript ". 4. In figure 3, the reference was pVir-CR-hvKP-RJ9299, and the pink ring represented pLVPK, the cyan ring represented pVir-CR-hvKP4, and the blue ring represented the pVir-CR-hvKP-RJ9299. However, the coverage of pVir-CR-hvKP-RJ9299 was not 100%. The analysis had a mistake, please correct it.
Sorry, it is a mistake. The reference was pLVPK. Please see the figure 3. We have corrected it.

5.
Only 15 SNPs at most existed between these isolates, but the virulence level was different. It will be innovative to find the reasons determining the diversity of virulence.
Yes, it is interesting that 15 SNPs at most existed between these isolates, but the virulence level was different. We will investigate the mechanism of virulence difference between these isolates.
Minor comments: 1. The authors identified the CR-hvKP with positive string test, so the first sentence "All eleven CR-hvKP outbreak isolates had a hypermucoviscous phenotype with positive string test" is not necessary.
Thank you for your close examination. We have corrected it. Please see the RESULTS section in "Marked-Up Manuscript ".
2. Figure 1: I cannot find "another two isolates were respectively from blood and drainage" in the figure 1. Please correct it.
RJ9337 was from drainage and RJ9690 was from blood. We have corrected it in the Figure 1. Furthermore, We have supplemented the clinical history of eleven patients in Table 1. There is also specimen source of the eleven isolates in Table 1. 3. Figure 2: "The eleven ST859-K19 CR-hvKP isolates were clustered into clade I. Six ST11-K64 KPC-2 producing isolates which had positive string test, namely CR-hvKP, were clustered into clade III. Nine ST11-K64 KPC-2 producing isolates which had negative string test, namely CRKP, were clustered into clade II." I think the figure 2A had a mistake, and please clarify it.
Thank you for your close examination. We have corrected it. The right is "ST11-K64 KPC-2 producing isolates which had positive string test, namely CR-hvKP, were clustered into clade II.
Nine ST11-K64 KPC-2 producing isolates which had negative string test, namely CRKP, were clustered into clade III." Please see the RESULTS section which marked in blue in "Marked-Up Manuscript ".
We have annotate the deletion region in figure 4B.

Reviewer #2
1. This is a description of an outbreak of K. pneumoniae ST859 (which is a single locus variant (SLV) of ST11) carrying a virulence plasmid and blaKPC-2, the latter in a separate plasmid to the former, among 11 patients, several of whom unfortunately died. For me, I find the insistence on this being novel because it is a new ST (it is an SLV of ST11, with probably only a single nucleotide change in the tonB gene) is overplayed and I would always exercise caution in describing an account as the first description.
Yes, it should be cautious for us to use the first time to describe ST859 (an SLV of ST11) isolates. We use the first time because we blasted tonB gene in ST859 and tonB gene in ST11 and found there were many nucleotide changes, but not a single nucleotide change, between the two tonB genes. The image below (left) showed nucleotide blast results of tonB genes between RJ9860 isolate (in our study) and a ST11 isolate. Furthermore, we have searched PubMed by keywords "Klebsiella pneumoniae and ST859", there was no literature published in PubMed. The image below (right) showed the PubMed search result. So I described ST859 isolates as the first description left: results of tonB genes between RJ9950 isolate and a ST11 isolate Right: PubMed search result by keywords "Klebsiella pneumoniae and ST859" 2. The other thing that really strikes me about this is that the virulence plasmid is so like pLVPK, which is a non-conjugative virulence plasmid associated with hypervirulent K2-CG43. So how did it get into this type? Usually, where you find virulence plasmids in 'high-risk' clones such as ST11, ST15, ST147 etc., they are hybrid virulence/resistance plasmids that have become conjugative through recombination (see for example Xie et al., 2020 A hybrid plasmid formed by recombination of a virulence plasmid and a resistance plasmid in Klebsiella pneumoniae. J Glob Antimicrob Resist 23:466-470), which is actually a more frightening prospect -with a non-conjugative plasmid the virulence plasmid is at least confined to that type. Maybe you could discuss this?
Yes, it is a very good advice for us to discuss how the non-conjugative virulence plasmid get into ST859 isolates. Regarding the question how a non-conjugative virulence plasmid was not confined to 'high-risk' clones such as ST11, ST15, ST147 etc, but get into ST859 clone, it is probably the non-conjugative virulence plasmid in ST859 isolates was mobilized by the conjugative plasmid, such as incompatibility group F (IncF), from the hvKP strain into ST859 CRKP strains or by employing intermediate E. coli strains according to Xu et al., 2021 Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae. However, this procession shuold be further validated. We have added this content in the Discussion section marked inpurple in "Marked-Up Manuscript ".
3. I worry about your defining isolates as hypervirulent on the basis of the string test -it is at best just an indication.
Yes, as a phenotypic detection method, string test is just an indication for hypervirulent isolates. In our study, hvKP was defined by aerobactin detection accoring to Zhang Y et al. 4. There is no mention of how you did the nanopore sequencing; also did you use the fully assembled sequence of RJ9299 as reference, or just the Illumina sequence? Is the sequence of RJ9299 on GenBank (or similar) -if not, it needs to be and an accession number provided.
Sorry, we used PacBio platform, not nanopore for RJ9299 sequencing. We have corrected it. this is our negligence for no mention of how we did the PacBio sequencing. We have added this content in the MATERIALS AND METHODS section marked in purple in "Marked-Up Manuscript ". We used the fully assembled sequence of RJ9299 as reference. The complete whole-genome sequences of eleven CR-hvKP isolates have been deposited in the GenBank database under BioProject accession no. PRJNA799444. Please see the Data availability section which marked in purple in "Marked-Up Manuscript ". 5. ST23, ST65, ST1797, ST43, ST231 and ST307' There are quite as few more -like ST147, ST15, ST383... The following review paper may be helpful to include Yang X, Dong N, Chan EW, Zhang R, Chen S. Carbapenem Resistance-Encoding and Virulence-Encoding Conjugative Plasmids in Klebsiella pneumoniae. Trends Microbiol. 2021 Jan;29(1):65-83.
Yes, it is a very good suggestion for us to refer to this review. We have supplemented ST147, ST15, ST383, ST268, ST595, ST375, ST48 in this section which marked in purple in "Marked-Up Manuscript ". 6. 'while ST258 was the main prevalent clone in North America, Latin America, and several European countries (6-9). ' Can't help but think that this is outdated and overplayed -it certainly is not my experience in the UK.
Yes, we have searched PubMed and found: ST11 clone is predominantly found in China and South America, ST258 is mostly reported in the United States, ST512 is endemic in Italy and Greece, ST147 is mainly reported in India and Tunisia. Please see this section which marked in purple in "Marked-Up Manuscript ". 7. All the patients received antimicrobial treatment, including carbapenem alone or in the combination with other alternative antibiotics when necessary' Why were patients treated with carbapenem alone for a carbapenemase producing organism?
In the case of severe infection, patients were empirically and prophylacticly treated with carbapenem before the CRKP isolates were cultured. Furthermore, for infection caused by CRKP which showed low level resistance to carbapenem, carbapenem in combination with other drugs is also a good therapy in the case of limited available antibiotics.
8. fosA is intrinsic in K. pneumoniae Sorry, because of the limited knowledge about fosA , we do not have a clear understanding about fosA is intrinsic in K. pneumoniae. We will be very grateful if respectable reviewer can provide us a reference article. 9. 'In terms of virulence genes, they all carried aerobactin (iucABCDiutA) and salmochelin (iroE) regulator of mucoid phenotype A (rmpA and rmpA2)' -but the virulence plasmid carried iroBCDN; surely that should be mentioned here. Not sure about this chromosomal iroE.
Sorry, it is a mistake. All the eleven isolates sequenced by Illumina carried aerobactin (iucABCDiutA), salmochelin (iroE) and regulator of mucoid phenotype A (rmpA and rmpA2). For RJ9299 sequenced by Illumina and PacBio, the chromosome carried iroE, not iroBCDN. There were no iroBCDN genes in RJ9299. We have corrected it. Please see this section which marked in blue in "Marked-Up Manuscript ".
10. Number of SNPs -please say how many bases were compared. 5901 core genome SNPs between the 11 ST859 outbreak isolates and 15 genomes of ST11 K. pneumoniae were identified and used to construct the maximum likelihood tree. We have added this content in RESULTS section which marked in purple in "Marked-Up Manuscript ".

Reviewer #3:
This study is the first to report the prevalence and dissemination of ST859 CR-hvKp strains. The characteristics of 11 CR-hvKp strains were analyzed by DNA sequencing, and genome-wide analysis was performed, which has important reference value for clinical anti-infection treatment and nosocomial infection control. I have some comments as follows. 1. The MIC data in Table 1 is the exact MIC value, or has the prefix "{less than or equal to}" or "{greater than or equal to}"? which is inconsistent with the description of the data in the text. In the text, it shows "{greater than or equal to}64", "{greater than or equal to}128" and "{less than or equal to} 0.25" etc. For example, not all 11 strains of imipenem have a MIC of "{greater than or equal to}64", and the MIC range should be 32-{greater than or equal to}128 mg/L according to table 1.
The MIC data in Table 1 is the exact MIC value. We have corrected this section accoring to the requirements of reviewer. Please see this section which marked in red in "Marked-Up Manuscript ".
2. Table 2 shows 12 strains of CR-hvKP, but in the text and Table 1 show 11 strains of CR-hvKP, with one more strain of RJ 10091. Resistance genes should also describe the presence or absence of plasmid-mediated quinolone and aminoglycoside resistance genes.
Thank you for your close examination. We have deleted the extra strain of RJ 10091 in Table 2. We have described the presence of plasmid-mediated quinolone and aminoglycoside resistance genes in text and in Table 2. Please see this section which marked in red in "Marked-Up Manuscript ".
3. This study needs to supplement the clinical history of 11 patients, including patient demographic data, isolation date, specimen source, antibiotic treatment, underlying diseases, and outcomes. The medical history data can also be preliminarily compared with the virulence test results to observe whether strains with strong in vitro virulence are more likely to cause the death of patients.
Yes, we have supplemented the clinical history of 11 patients in Table 1  Thank you for submitting your revised manuscript to mSystems. I have completed the review and discussed with a senior editor. We concluded that you and your co-authors have adequately addressed most of the previous reviewer comments. However, the reviewers have raised new issues (see details below). One major concern is that description of details for the methods is lacking. Please address these issues carefully in your next revision.
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